WO2024014643A1 - Procédé de production d'immunocytes présentant une cytotoxicité améliorée - Google Patents

Procédé de production d'immunocytes présentant une cytotoxicité améliorée Download PDF

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WO2024014643A1
WO2024014643A1 PCT/KR2023/002414 KR2023002414W WO2024014643A1 WO 2024014643 A1 WO2024014643 A1 WO 2024014643A1 KR 2023002414 W KR2023002414 W KR 2023002414W WO 2024014643 A1 WO2024014643 A1 WO 2024014643A1
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antibody
human immunoglobulin
aldesleukin
plasma
cells
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PCT/KR2023/002414
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Korean (ko)
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문귀영
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주식회사 노보셀바이오
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • the present invention relates to a method for producing immune cells with improved cytotoxicity.
  • lymphocytes Natural Killer cells
  • NK immune cells are activated and cultured using animal-derived antibody reagents.
  • animal-derived antibody reagents In order to create a cell therapy product that can be administered to humans, animal-derived antibody reagents must be excluded. Since most antibody reagents are products that can be used only for research purposes and are not suitable for use in the production of cell therapy for administration to humans, there has been a continuous demand to date.
  • the present inventors completed the present invention by confirming that better efficacy can be achieved by activating NK cells using medicines produced in a GMP facility.
  • One aspect of the present invention is the process of (a) adding any one or more of Aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma to lymphocytes and culturing them once to Repeat 7 times; (b) In the process of culturing any one or more of the above repeated steps, Panobinostat, Trichostatin A, valproic acid, sodium butyrate, and SAHA (suberoylanilide hydroxamic acid) ) further adding any one or more of; and (c) harvesting the cultured lymphocytes.
  • the purpose is to provide a method for producing immune cells with improved cytotoxicity.
  • Another aspect of the present invention is (a) in the culture of lymphocytes, 1 adding any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, 2 panobinostat, trichostatin A, adding at least one of valproic acid, sodium butyrate, and SAHA; and (b) harvesting the lymphocytes cultured in step (a).
  • the purpose is to provide a method for producing immune cells with improved cytotoxicity.
  • One aspect of the present invention is the process of (a) adding any one or more of Aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma to lymphocytes and culturing them once to Repeat 7 times; (b) In the process of culturing any one or more of the above repeated steps, Panobinostat, Trichostatin A, valproic acid, sodium butyrate, and SAHA (suberoylanilide hydroxamic acid) ) further adding any one or more of the following; and (c) harvesting the cultured lymphocytes.
  • step (a) may involve adding at least one of anti-CD56 antibody and human immunoglobulin and at least one of aldesleukin, IL-15, IL-18, and plasma. .
  • 0.5 to 5 ⁇ l of the anti-CD56 antibody and 5 to 500 ⁇ l of human immunoglobulin may be added.
  • the valproic acid may be added at a concentration of 50 to 5000 uM.
  • Another aspect of the present invention is (a) adding any one or more of the following to the culture of lymphocytes; and 1 adding any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; 2 Addition of any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA; (b) harvesting the lymphocytes cultured in step (a); providing a method for producing immune cells with improved cytotoxicity, including:
  • step (a) there may be a method for producing immune cells with improved cytotoxicity, further comprising step (a-1) in which one or more of the following is added to lymphocytes; 1 Addition of any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; 2 Add one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • step (a-1) there may be a method for producing immune cells with improved cytotoxicity, further comprising step (a-2) in which one or more of the following is added to lymphocytes; 1 Addition of any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; 2 Add one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • step (a-2) there may be a method for producing immune cells with improved cytotoxicity, further comprising step (a-3) in which one or more of the following is added to lymphocytes; 1 Addition of any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; 2 Add one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • step (a-3) there may be a method for producing immune cells with improved cytotoxicity, further comprising step (a-4) in which one or more of the following is added to lymphocytes; 1 Addition of any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; 2 Add one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • step (a-4) there may be a method for producing immune cells with improved cytotoxicity, further comprising step (a-5) in which one or more of the following is added to lymphocytes; 1 Addition of any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; 2 Add one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma are added to at least one of anti-CD56 antibody and human immunoglobulin. It may be processing any one or more of aldesleukin, IL-15, IL-18, and plasma.
  • 0.5 to 5 ⁇ l of the anti-CD56 antibody and 5 to 500 ⁇ l of human immunoglobulin may be added.
  • the valproic acid may be added at a concentration of 50 to 5000 uM.
  • the production method of the present invention can provide immune cells with improved cytotoxicity.
  • Figures 1 to 4 show the results of confirming the ratio of NK and NKT cells produced by the production method according to an embodiment of the present invention.
  • Figures 5 and 6 show the results of confirming the receptor expression level of NK cells produced by the production method according to one embodiment of the present invention.
  • Figures 7 to 12 show the results of tests to confirm the toxicity of NK cells produced by the production method according to one embodiment of the present invention against cancer cells.
  • Figures 13 to 16 show the results of a test to confirm the secretion amount of Interferon gamma (IFN-r) from NK cells produced by the production method according to an embodiment of the present invention.
  • IFN-r Interferon gamma
  • One aspect of the present invention is the process of (a) adding any one or more of Aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma to lymphocytes and culturing them once to Repeat 7 times; (b) In the process of culturing any one or more of the above repeated steps, Panobinostat, Trichostatin A, valproic acid, sodium butyrate) and SAHA (suberoylanilide hydroxamic acid) Adding one or more of acid); and (c) harvesting the cultured lymphocytes. It provides a method for producing immune cells with improved cytotoxicity.
  • the repeating step is a step of repeating the treatment of lymphocytes with one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma 1 to 7 times.
  • Repetition of the above steps means repetition of processing of any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma.
  • the preparations processed in the repeated steps are processed in each step. may be different from each other.
  • the human immunoglobulin is not coated on the flask, but is treated directly on the lymphocytes during the culture process. do. This can simplify the process and shorten the time required for coating.
  • 'lymphocytes are the central cells that constitute the immune system and have the characteristics of adaptive immunity, antigen specificity, receptor diversity, memory, and self-non-self distinction. They play a central role in adaptive immunity by recognizing antigens presented by different types of white blood cells after phagocytosing foreign substances and secreting cytokines and antibodies through them.
  • the lymphocytes may include NK cells.
  • the lymphocytes may be obtained by centrifugation after separation from peripheral blood, or may be prepared by suspending them in a buffer solution, etc. for additional procedures.
  • the treatment of any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma is performed with one or more of anti-CD56 antibody and human immunoglobulin. Any one or more of aldesleukin, IL-15, IL-18, and plasma may be added. According to one embodiment of the present invention, it can be confirmed that the survival rate and cytotoxicity of NK cells are significantly improved through this treatment.
  • the amount of anti-CD56 antibody used may be 0.5 to 5 ⁇ l for the anti-CD56 antibody, and preferably 5 to 500 ⁇ l for human immunoglobulin, more preferably 5 to 50 ⁇ l. You can. If treated outside the above content range, the cells are not activated or the cells die, making it impossible to obtain the improved cytotoxicity or cell viability effects that are intended to be achieved in the present invention.
  • the step further comprising treatment with any one or more of the panobinostat, trichostatin A, valproic acid, sodium butyrate, and suberoylanilide hydroxamic acid (SAHA) is repeated.
  • steps 1 to 1 of further treating with one or more of the histone deacetylase (HDAC) inhibitors panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA is repeated.
  • HDAC histone deacetylase
  • the above-mentioned lymphocytes are treated with panobinostat, trichotate between repetitions or after 1 to 7 repetitions of treatment of any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma.
  • panobinostat trichotate between repetitions or after 1 to 7 repetitions of treatment of any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma.
  • the lymphocytes are additionally processed after processing one or more of antibodies, human immunoglobulins, and plasma, any of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulins, and plasma are added to the lymphocytes. It may be processed 1 to 7 times at any point between or after one or more treatment repetitions. When additional processing is repeated, the additionally processed preparations may be the same or different at each step.
  • the valproic acid may be treated at a concentration of 50 to 5000 uM. If treated outside the above content range, the effect of improved cytotoxicity or cell viability desired in the present invention cannot be obtained. According to one embodiment, when valproic acid is less than 50 uM, cytotoxicity or cell viability effects do not occur, and when valproic acid is more than 5000 uM, cell proliferation does not occur.
  • Another aspect of the present invention is (a) in the culture of lymphocytes, 1 adding any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, 2 panobinostat, trichostatin A, adding at least one of valproic acid, sodium butyrate, and SAHA; and (b) harvesting the lymphocytes cultured in step (a). It provides a method for producing immune cells with improved cytotoxicity.
  • lymphocytes are treated with any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or aldesleukin, IL-15, IL-18, anti -It may be processing any two or more of CD56 antibody, human immunoglobulin, and plasma, and further processing any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • it may further include a step (a-1) in which any one or more of the following treatments are performed on the treated lymphocytes after step (a) but before step (b); 1 Addition of any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; 2 Addition of one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • step (a-1) the lymphocytes treated in step (a) are treated with one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or panobinostat. , trichostatin A, valproic acid, sodium butyrate, and SAHA may be further treated, or all of these treatments may be performed.
  • aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma may be further processed with any one or more of anti-CD56 antibody, human immunoglobulin, and plasma, or may be treated with any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA, (a )
  • any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma are treated, and any of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA is treated.
  • any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma is further processed, followed by panobinostat, trichostatin A, valproic acid, It may be processing any one or more of sodium butyrate and SAHA.
  • a step (a-2) in which any one or more of the following treatments are performed on the treated lymphocytes after step (a-1) and before step b) may be further included; 1 Addition of any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; 2 Addition of one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • step (a-2) the lymphocytes treated in step (a-1) are treated with any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or It may be further treated with one or more of vinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA, or all of these treatments may be performed.
  • aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma may be further processed with any two or more of anti-CD56 antibody, human immunoglobulin, and plasma, or any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA, a -If any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA were treated in step 2), aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin and plasma may be further processed and treated with any one or more of panobinostat, trichostatin A, valproic acid, and sodium butyrate.
  • One embodiment of the present invention may further include a step (a-3) in which any one or more of the following treatments are performed on the treated lymphocytes after step (a-2) and before step (b); Treatment with any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; Treatment with any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • the lymphocytes treated in step (a-2) are treated with one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or It may be further treated with one or more of vinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA, or all of these treatments may be performed.
  • aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma are treated in step (a-2), aldesleukin, IL-15, It may be further processed with any one or more of IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA,
  • step (a-2) any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma are treated, followed by panobinostat, trichostatin A, valproic acid, and sodium butyrate.
  • aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma are further processed, panobinostat, trichostatin A, It may be processing any one or more of valproic acid, sodium butyrate, and SAHA.
  • One embodiment of the present invention may further include a step (a-4) in which any one or more of the following treatments are performed on the treated lymphocytes after step (a-3) and before step (b); Treatment with any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; Treatment with any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • step (a-4) the lymphocytes treated in step (a-3) are treated with any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or It may be further treated with one or more of vinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA, or all of these treatments may be performed.
  • aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma aldesleukin, IL-15, It may be further treated with any two or more of IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA, If any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA is treated in step (a-3), aldesleukin, IL-15, IL-18, anti-CD56 antibody, human Any one or more of immunoglobulins and plasma may be further processed and any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA may be processed.
  • a step (a-5) of subjecting the treated lymphocytes to one or more of the following treatments after step (a-4) and before step (b) may be further included; Treatment with any one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma; Treatment with any one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA.
  • the lymphocytes treated in step (a-4) are treated with one or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or It may be further treated with one or more of vinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA, or all of these treatments may be performed.
  • aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma are treated in step (a-4), aldesleukin, IL-15, It may be further processed with any one or more of IL-18, anti-CD56 antibody, human immunoglobulin, and plasma, or one or more of panobinostat, trichostatin A, valproic acid, sodium butyrate, and SAHA,
  • step (a-4) any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma are treated, followed by panobinostat, trichostatin A, valproic acid, and sodium butyrate.
  • aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma are further processed, panobinostat, trichostatin A, It may be processing any one or more of valproic acid, sodium butyrate, and SAHA.
  • the treatment of any two or more of aldesleukin, IL-15, IL-18, anti-CD56 antibody, human immunoglobulin, and plasma is performed with any one or more of anti-CD56 antibody and human immunoglobulin. It may be processing any one or more of aldesleukin, IL-15, IL-18, and plasma. Specifically, the main ingredient may be one or both of anti-CD56 antibody and human immunoglobulin, while also treating one or more of aldesleukin, IL-15, IL-18, and plasma. According to one embodiment of the present invention, this treatment can improve the viability and cytotoxicity of NK cells.
  • the amount of anti-CD56 antibody used may be 0.5 to 5 ⁇ l, and the amount of human immunoglobulin used may be preferably 5 to 500 ⁇ l, more preferably 5 to 50 ⁇ l.
  • the valproic acid may be treated at a concentration of 50 to 5000 uM.
  • cells in the step of harvesting cultured lymphocytes, cells can be obtained by a known method, and specifically, only the desired lymphocytes can be separated and harvested through centrifugation. Immune cells produced through this manufacturing method have significantly improved cytotoxicity compared to conventional methods and can be used as various cell therapeutic agents.
  • immune cells containing NK cells with improved cytotoxicity were prepared through a total of 9 steps as follows.
  • Lymphocytes were isolated from human peripheral blood. Specifically, 60 ml to 90 ml of human peripheral blood was collected using a heparinized 10 ml vacuum collection tube (BD Vacutainer TM). Afterwards, 15 ml of Ficoll-Paque Plus (endotoxin tested, density 1.077 g/ml, GE Healthcare, USA) solution was added to a 50 ml lymphocyte separation tube (Leuco sep, Greiner Bio-One, Swiss), and 2,000 By centrifugation at rpm, the solution settled to the bottom of the glass membrane in the tube.
  • BD Vacutainer TM heparinized 10 ml vacuum collection tube
  • Ficoll-Paque Plus endotoxin tested, density 1.077 g/ml, GE Healthcare, USA
  • the collected blood was transferred to a separation tube and centrifuged at 2,500 to 3,500 rpm for 12 to 25 minutes to separate the red blood cells and granulocytes into the lower layer and the monocyte layer (lymphocyte layer), platelets, and plasma into the upper layer. Afterwards, the upper layer of plasma was inactivated in a 56°C water bath for 30 minutes.
  • the lymphocyte layer was collected with a sterilized pipette, collected in a 15 ml tube, and then centrifuged to remove the supernatant. Lymphocytes from which the supernatant was removed were suspended in 10 ml of buffer solution (PBS) and washed. The cell count of a portion of the suspension was measured using a hemocytometer, and then centrifuged again to collect only lymphocytes. The total number of harvested lymphocytes was measured to be 38 x 10 6 .
  • the lymphocytes isolated in the first step were suspended in culture medium (KBM 502) and cultured using two 25T flasks (Flask A, Flask B) (hereinafter, culture in Flask A is referred to as culture method A and Flask B). Culture is referred to as culture method B). 50 to 300 ⁇ l of Aldesleukin, IL-18, and human immunoglobulin were added to the cell suspension in Flask A and Flask B, respectively, and then incubated in a 37°C 5% CO 2 incubator for 1 to 2 days. Immune cells were cultured. Antibodies and plasma in this step were added to the suspension on the day the culture began.
  • the cultured cells were transferred to a 75T flask and the immune cells were cultured in an incubator at 37°C and 5% CO 2 for 2 to 3 days.
  • 50 to 300 ⁇ l of aldesleukin, IL-18, and human immunoglobulin were added to the cell suspension in Flask A and Flask B, respectively, and then immunized in a 75T Flask for 1 to 2 days at 37°C in a 5% CO 2 incubator.
  • Cells were cultured.
  • Antibodies and plasma in this step were added to the suspension on the day the culture began.
  • New medium was added to each suspension of Flask A and Flask B in the fourth step, where immune cells were cultured, and then aldesleukin, IL-18, and human immunoglobulin were added in the same manner as in the fourth step. .
  • the cultured cells were transferred to a 175T flask and the immune cells were cultured in an incubator at 37°C and 5% CO 2 for 2 to 3 days.
  • 50 to 300 ⁇ l of aldesleukin, IL-18, and human immunoglobulin were added to the cell suspension in Flask A and Flask B, respectively, and Panobinostat, Trichostatin A, One or more of valproic acid, sodium butyrate, and suberoylanilide hydroxamic acid (SAHA) was treated to a concentration of 100 to 500 uM.
  • SAHA suberoylanilide hydroxamic acid
  • Step 7 Addition/non-addition of NK cell activating substance
  • the cell suspensions cultured in Flask A and Flask B in step 7 were transferred to 1,000 ml CO 2 permeable bags (Bag A, Bag B), respectively, and incubated in an incubator at 37°C and 5% CO 2 for 7 to 10 days. Immune cells were cultured. Plasma in this step was added on the 7th day of culture. Also, at this time, at least one of Panobinostat, Trichostatin A, valproic acid, sodium butyrate, and SAHA (suberoylanilide hydroxamic acid) was added to Bag B. .
  • the cell suspensions according to culture method A and culture method B contained in the CO 2 permeable bag were transferred to a centrifuge tube and then centrifuged at 2,500 rpm to harvest the cells. The supernatant was discarded, the pellet containing cells was washed twice with 250 ml of sterile saline solution, and the cells were harvested by centrifugation. The harvested cells were injected into a 100 ml pack of sterile physiological saline for injection to complete the production of immune cells containing NK cells with improved cytotoxicity.
  • NK and NKT In order to confirm the cell ratio of NK and NKT according to the present invention, 1x10 6 /ml of non-treated NK cells cultured for 14 days with Valproic acid, Trichostatin A, SAHA, and Panobinostat were taken, and CD3-APC, CD56-FITC, 0.5 ul of CD16-PE-Cy7 was added and the cells were stained for 30 minutes at room temperature, shielded from light. After the staining was completed, the cells were centrifuged at 1,200 rpm at 4°C for 3 minutes to remove the supernatant. The cells were suspended in PBS and the ratio of NK and NKT cells was observed using flow cytometry.
  • the Trichostatin A treated group increased the NK cell ratio by 3% and the NKT cell ratio by 1.4% compared to the untreated group, and the NK cell ratio of the SAHA treated group increased by 1.4% compared to the untreated group. It was observed that the NKT cell ratio was significantly increased to 11.6% and 2.9%.
  • panobinostat-treated group was found to have a significantly increased NK cell rate of 11.6% and NKT cell rate of 1.32% compared to the non-treated group.
  • NK cells proliferated by adding Valproic acid, Trichostatin A, SAHA, and Panobinostat could increase the ratio of NK and NKT cells compared to the control group.
  • NK cells cultured for 14 days were taken and analyzed for CD3-APC, CD56-FITC, and CD16-PE.
  • -Cy7, NKG2D-PE, and DNAM-1-Percp-Cy5.5 were added in 0.5 ul each, blocked from light, and the cells were stained for 30 minutes at room temperature. After staining was completed, the cells were centrifuged at 1,200 rpm at 4°C for 3 minutes to remove the supernatant. The cells were suspended in PBS and the expression levels of DNAM-1 and NKG2D were confirmed by flow cytometry.
  • Lung cancer cells A549), breast cancer cells (MDA-MB231), blood cancer cells (K562), epidermal cancer cells (A431), pancreatic cancer cells (Panc-1), and liver cancer cells (SK-Hep1) stained with Calcein-AM.
  • % FBS in phenol red free RPMI media was homogenized and dispensed into a v-bottom 96 well plate at 1x10 4 /well.
  • the cultured NK cells were added at 40 or 100 times the number of cancer cells, and the NK cells and cancer cells were co-cultured in an incubator at 37°C with 5% CO 2 for 6 hours.
  • the blood cancer cell line (K562) increased by 44%
  • the lung cancer cell line (A549) increased by 26%
  • the breast cancer cell line (MDA-MB231) increased by 44% compared to the untreated group.
  • 17% increased cytotoxicity was observed in the liver cancer cell line (SK-Hep1).
  • the SAHA-treated group had 46% more cells in the blood cancer cell line (K562), 26% more cells in the lung cancer cell line (A549), and breast cancer cell line (MDA) than the untreated group. Increased cytotoxicity of 18% was observed in the epidermal cancer cell line (A431), 39% in the epidermal cancer cell line (A431), and 74% in the pancreatic cancer cell line (Panc-1).
  • the lung cancer cell line (A549) increased by 33%
  • the breast cancer cell line (MDA-MB231) increased by 24%
  • the liver cancer cell line (SK-Hep1) increased by 18% compared to the non-treated group. % increased cytotoxicity was observed.
  • NK cells cultured with Valproic acid, Trichostatin A, SAHA, and Panobinostat increase cytotoxicity against most solid cancers and blood cancers compared to the control group.
  • Experiment example 4 Test to confirm the secretion amount of Interferon gamma (IFN-r) from NK cells
  • NK cells and cancer cells cultured for 14 days were co-cultured in 96 wells for 6 hours.
  • the method for measuring IFN-r followed the instructions of the Human IFN-gamma quantikine ELISA Kit from RnD Systems.
  • NK cells cultured with Valproic acid, Trichostatn A, SAHA, and Panobinostat had a cytotoxic effect on most solid cancer and blood cancer cells when compared to the control group.

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Abstract

La présente invention concerne un procédé de production d'immunocytes présentant une cytotoxicité améliorée. Le procédé de production peut permettre d'obtenir des immunocytes présentant une cytotoxicité améliorée, et les immunocytes peuvent être utilisés comme divers produits de thérapie cellulaire selon la cytotoxicité améliorée.
PCT/KR2023/002414 2022-07-11 2023-02-20 Procédé de production d'immunocytes présentant une cytotoxicité améliorée WO2024014643A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2022-0085308 2022-07-11
KR20220085308 2022-07-11
KR1020230011635A KR20240008233A (ko) 2022-07-11 2023-01-30 세포 독성이 향상된 면역세포 제조방법
KR10-2023-0011635 2023-01-30

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WO2024014643A1 true WO2024014643A1 (fr) 2024-01-18

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170007692A (ko) * 2015-07-10 2017-01-19 고려대학교 산학협력단 자연 살해세포의 대량증식 방법 및 배양용 조성물
KR20190060412A (ko) * 2017-11-24 2019-06-03 의료법인 성광의료재단 Nk 배양용 조성물 및 이를 이용하여 nk 세포를 배양하는 방법
KR20190131239A (ko) * 2018-05-16 2019-11-26 고려대학교 산학협력단 Hdac 억제제를 이용한 사람 유래 자연살해세포의 확장 배양법
KR20200061240A (ko) * 2018-11-23 2020-06-02 차의과학대학교 산학협력단 자연살해세포의 활성 증진용 조성물 및 이의 용도
KR20220036287A (ko) * 2020-09-15 2022-03-22 주식회사 티에스바이오 고순도 및 고효율의 자연살해세포 제조방법 및 이의 용도

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170007692A (ko) * 2015-07-10 2017-01-19 고려대학교 산학협력단 자연 살해세포의 대량증식 방법 및 배양용 조성물
KR20190060412A (ko) * 2017-11-24 2019-06-03 의료법인 성광의료재단 Nk 배양용 조성물 및 이를 이용하여 nk 세포를 배양하는 방법
KR20190131239A (ko) * 2018-05-16 2019-11-26 고려대학교 산학협력단 Hdac 억제제를 이용한 사람 유래 자연살해세포의 확장 배양법
KR20200061240A (ko) * 2018-11-23 2020-06-02 차의과학대학교 산학협력단 자연살해세포의 활성 증진용 조성물 및 이의 용도
KR20220036287A (ko) * 2020-09-15 2022-03-22 주식회사 티에스바이오 고순도 및 고효율의 자연살해세포 제조방법 및 이의 용도

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